Plants produce a vast and diverse array of low-molecular-weight organic compounds. A small number of these are primary metabolites, which are common to all plant species as they are directly required for growth and development. The remaining, overwhelming majority of these natural products are considered secondary metabolites and are not found in all plants. Thus, individual species produce a limited subset of all plant natural products, although families will sometimes share common secondary metabolism (e.g., oleoresinosis in Pinaceae). Nevertheless, many secondary metabolites have important ecological roles, particularly in plant defense. (Croteau et al. 2000). For example, phytoalexins are produced in response to microbial infections and exhibit antimicrobial properties (VanEtten et al. 1994), while allelochemicals are secreted to the rhizosphere, and suppress germination and growth of neighboring seeds (Bais et al. 2004).
Particularly abundant in plants, as both primary and secondary metabolites, are terpenoids, which comprise the largest class of natural products and exhibit wide diversity in chemical structure and biological function (Croteau et al. 2000). Much of the structural variation within this class arises from the diverse carbon backbones formed by terpene synthases (cyclases). These divalent metal ion dependent enzymes carry out complex electrophilic cyclizations and/or rearrangements to create these diverse skeletal structures from relatively simple acyclic precursors (Davis and Croteau 2000). Notably, production of a specific backbone structure either dictates, or at least severely restricts, the metabolic fate of that particular molecule. Thus, terpenoid biosynthesis is often controlled, at least in part, by regulating terpene synthase activity [e.g. giberellin biosynthesis; (Silverstone et al. 1997)].
A substantial fraction of the known terpenoids can be classified as labdane-related diterpenoids (20 carbon). These are defined here as minimally containing the bicyclic hydrocarbon structure found in the labdane class of diterpenoids, although this core structure can be further cyclized and rearranged, as in the related/derived structural classes (e.g., kauranes, abietanes, and [iso]pimaranes). Significantly, this includes the primary metabolite gibberellin growth hormones. However, the vast majority of the more than 5,000 known labdane-related diterpenoids are secondary metabolites.
Biosynthesis of labdane-related diterpenoids is initiated by class II terpene synthases which catalyze formation of the characteristic bicyclic backbone in producing specific stereoisomers of labdadienyl/copalyl disphosphate (CPP) from the universal diterpenoid precursor, and plant primary metabolite, (E,E,E)-geranylgeranyl diphosphonate (GGPP). In addition, this core bicyclic structure is always further modified by stereoselective CPP specific class I terpene synthases (i.e. ionization of the diphosphate moiety to form one or more new carbon-carbon bonds). Thus, class II and class I terpene synthases act sequentially in catalyzing stereochemically coupled cyclization reactions to form labdane-related diterpene skeletal backbones.
Significantly, the class II protonation-initiated bicyclization reaction is fundamentally different than the diphosphate ionization initiated reactions catalyzed by the more common class I terpene synthases. Nevertheless, the class II cyclases clearly fall within the terpene synthase gene family (Bohlmann et al. 1998b). However, rather than the DDXXD metal binding motif functionally associated with class I activity (Davis and Croteau 2000), class II terpene cyclases contain a distinct DXDD motif (Sun and Kamiya 1994) which has been functionally associated with class II cyclization reactions (Peters et al. 2001).
Prototypical plant class I terpene synthases are similar in size and contain two structurally defined domains (Starks et al. 1997; Whittington et al. 2002). However, some terpene synthases, and in particular all of those involved in labdane-related diterpenoid biosynthesis, contain a large amount of additional amino terminal sequence termed the ‘insertional’ element [approximately 240 amino acid residues; (Peters and Croteau 2002)]. Notably, given adequate sequence information, this specific structural feature is useful for putative identification of labdane-related diterpene synthases, although it is not sufficient for even such generalized functional annotation [e.g. (Bohlmann et al. 1998a)].
Rice (Oryza sativa) provides a model system to investigate labdane-related diterpenoid biosynthesis, as this well characterized plant is known to produce a number of such natural products beyond the ubiquitous gibberellic acid (GA) growth hormones (FIG. 1). These compounds include momilactones A and B (Kato et al. 1973; Cartwright et al. 1981), oryzalexins A to F (Akatsuka et al. 1985; Sekido et al. 1986; Kato et al. 1993; 1994), oryzalexin S (Kodama et al. 1992), and phytocassanes A to E (Koga et al. 1995; Koga et al. 1997). All of these natural products are produced in leaves in response to infection with the blast pathogenic fungus Magneportha grisea and exhibit antimicrobial properties; thus qualifying as phytoalexins (VanEtten et al. 1994). In addition, momilactones A and B also act as allelochemicals, as these were originally identified as dormancy factors from rice seed husks (Kato et al. 1973), and momilactone B has recently been shown to be constitutively secreted from the roots of rice seedlings, where it acts as an allelopathic agent (Kato-Noguchi and Ino 2003). Further, secretion of antimicrobial agents to the rhizosphere may also provide a competitive advantage for root establishment through local suppression of soil micro-organisms (Bais et al. 2004).
Conveniently, rice leaves produce all of these secondary metabolites after UV irradiation as well as blast fungal infection (Kodama et al. 1988), providing a standard method for inducing biosynthesis of these natural products and, presumably, transcription of the corresponding enzymatic machinery. In particular, it has previously been shown that UV irradiation induces biosynthesis of ent-sandaracopimaradiene, syn-pimara-7,15-diene, and syn-stemar-13-ene, the putative precursors to oryzalexins A to F, momilactones A and B, and oryzalexin S, respectively (Wickham and West 1992). These polycyclic diterpene hydrocarbons further have been demonstrated to be selectively produced via CPP of the corresponding stereochemistry [i.e. ent or syn; (Mohan et al. 1996)]. More recent work has identified the class I diterpene synthase producing ent-cassa-12,15-diene, the putative precursor to phytocassanes A to E (Yajima et al. 2004), stereoselectively from ent-CPP (Cho et al. 2004). In addition, it was also recently reported that only a single CPP synthase gene (OsCPS1) is involved in GA biosynthesis, although no sequence information was presented (Sakamoto et al. 2004). Thus, gene function was demonstrated by the severe growth defect (i.e. dwarf phenotype) of the corresponding mutant (i.e. T-DNA insertion) plant, along with its rescue by exogenous application of GA3. Finally, although other putative class II and class I labdane-related diterpene synthase genes can be found in the rice genome, gene isolation and biochemical characterization have not been previously reported, leaving in question the role and specific activity of these additional cyclases.
It is therefore a primary objective of the present invention to identify, isolate and purify nucleic acid fragments encoding class II terpene synthases.
It is a further objective of the present invention to identify, isolate, and purify a nucleic acid fragment encoding a syn-copalyl diphosphate synthase.
It is still a further objective of the present invention to identify, isolate, and purify a nucleic acid fragment encoding a syn-copalyl diphosphate specific 9β-pimara-7,15-diene synthase.
It is a further objective of the present invention to identify, isolate, and purify a nucleic acid fragment encoding ent-copalyl diphosphate synthases.
It is a further objective of the present invention to provide a method of modulating terpenoid biosynthesis.
It is a further objective of the present invention to provide a method of modulating expression of class II terpene synthases.
It is yet a further objective of the present invention to provide a method of modulating expression of a syn-copalyl disphosphate.
It is a further objective of the present invention to provide a method of modulating expression of syn-copalyl diphosphate specific pimara-7,15-diene synthase.
It is a further objective of the present invention to provide a method of modulating expression of ent-copalyl diphosphate synthases.
The method and means of accomplishing each of the above objectives as well as others will become apparent from the detailed description of the invention which follows hereafter.